Name: a miniaturized glycan microarray assay for assessing avidity and specificity of influenza a virus hemagglutinins
Uploaded: 2016-05-29T14:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

这项工作由斯克里普斯芯片核心设施，并从疾病控制中心（JCP）合同的一部分得到了支持。 RPdV是来自荷兰科学研究组织（NWO）一个人Rubicon和VENI授权的收件人。由NIGMS授予GM62116（JCP）资助的财团为功能糖组学（http://www.functionalglycomics.org/）提供在这项研究中使用的几个聚糖。这是来自斯克里普斯研究所的出版物29113。

注:所有步骤都在室温下进行，除非另有说明。 1.阵列建设<ol><li>多糖制备及板设置<ol><li>准备打印缓冲区的股票。首先使500毫升磷酸氢钠的150mM的储备液。也使50毫升磷酸二氢钠溶液的150毫原液。在烧瓶中，使用磁力搅拌棒和pH计，直到达到pH为8.5缓慢滴定与一元溶液的磷酸氢钠溶液中。添加吐温-20〜0.005％。 </li><li>准备聚糖样品。稀释PAA结合聚糖（diLacNAc（Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA），3SLNLN（Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA）和6SLNLN（Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA（ 图1A ））被以稀释至100μG/ ml，在从step1.1.1。单价聚糖打印缓冲器，diLacNAc（Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-（CH 2）3 NH 2，3SLNLN（Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ - （CH 2）3 NH 2）和6SLNLN（Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1- 4GlcNAcβ - （CH 2）3 NH 2）），以100μM在从步骤1.1.1打印缓冲器。最后，准备胺官能染料，如方格标记/着陆灯使用。染料标记点是在1μM打印。 注:Atto488-NHS染料中的打印使用。然而，任何胺官能染料，由滑动扫描器可读适合此目的。 </li><li>转移10微升每聚糖样品至384孔微量滴定板的，用于印刷的机器人。将未使用的聚糖在在密封管-20°C的打印缓冲溶液。 Tansfer 10微升染料标记印刷板。 </li></ol></li><li>印花 注意:所有STEps的这种担心触摸或移动必须用手套来完成的幻灯片。 <ol><li>根据布局的正确配置放置样仪销在打印头。此布局使用一个针打印所有样品和阵列。打印聚糖六块，跳过一个特征（特征被定义为某种化合物的单点），在每一侧，以使得相同样品的斑点不完全打印彼此旁边。 注:最终用户必须决定的配置。 </li><li>从袋，打开箱子除去幻灯片，并除去任何灰尘颗粒或小片的塑料，可能通过在每个滑动吹超高纯度氮气是在其表面上。放置幻灯片的期望数量，涂布面朝上，在点样仪阶段。 </li><li>节目的点样仪，使用的每次访问27点参数的源板，用3"预点"到聚-L-赖氨酸涂覆的载玻片，在打印针的尖端，以除去过量的液体<ol><li>打开MPU（主电源单元）和气候控制单元。打开计算机上平铺分析软件。 </li><li>选择布局打印。选择要用于打印阵列1×1的打印工具选项→针工具 - 1单针。选择目标选项卡→工具数组定义→打印模式与添加的间距（点到点的距离），将要打印的行和容纳样品的列的数量，以及样品的印刷图案（此阵列的8 ×8印刷图案在340微米间距为7总样本）中使用。 </li><li>选择目标→幻灯片版式和编程块到块的距离，让每个重复阵列打印到48孔（4×12）幻灯片模板。选择源和（当使用1针1对每个样品）（此打印7来源皮卡将被用来容纳6样品孔和1个染料标记井）添加源皮卡的数量。 注:源对话应该把绿色，以显示该号码源的访问匹配的印刷图案要打印的样品。 </li><li>选择目标→适配器板和幻灯片布局和调整幻灯片编号（5张幻灯片都是一次打印此打印1预去斑幻灯片）。 </li><li>通过点击T1按钮来激活托盘和添加幻灯片的所需数量的托盘，密切关注点样前滑（蓝色）和"真实的"幻灯片的位置释放托盘。 </li><li>广场上所有剩余的真空端口平原载玻片阻止真空，然后单击确定以激活真空。检查所有幻灯片就位，然后单击确定托盘返回初始位置。 </li><li>装载印版到所述板夹持器，并确保齿条来代替设置正确。点击GO，让打印过程继续进行。 </li></ol></li><li>打印中，每个阵列6点重复其余24个点（4阵列/源的访问）。 </li><li>加载384孔板装入打印机，开始打印。保持相对湿度，在打印时，在整个打印之间55和65％。打印5张幻灯片后，打印机停止。 注意:虽然在此测定法与微阵列中的特定的8×8图案被印刷，不同的微阵列印刷机器人将需要设备特定的编程来实现所需的布局。它是由最终用户来确定给定的其设备的规格的最适当的模式。 </li></ol></li><li>加湿/固定化 注意:一旦幻灯片打印结束后，他们接受一个临时的固定化步骤，也称为加湿。加湿印刷后有助于均匀通过重新润湿斑点，并确保完全阻断样品附件。 <ol><li>放于100％湿度室玻片一段30分钟印刷后立即。通过将非常湿纸巾平在一个大的玻璃盘的底部，放置幻灯片构造腔室关于某种架上，诸如试管架，打印面朝上的，并用保鲜膜套住中的水分密封。 </li><li>数用酒精/耐溶剂标志着一个干燥盒笔和存储幻灯片。变干过夜。 </li></ol></li><li>编号，阻塞和存储<ol><li>准备封闭液 - 50毫米乙醇胺在50 mM的硼酸盐缓冲液。首先，溶解硼酸，在DDH 2 O至50mM的终浓度在含有磁搅拌棒的烧瓶中。大力搅拌溶液在搅拌盘，直到所有的固体溶解。同时不断搅拌，从16.54 1M贮存液中添加乙醇胺适量以达到预期的50mM的浓度。添加浓氢氧化钠调整到9.2的最终pH。 </li><li>浸入印刷载玻片在1小时的封闭缓冲液溶液。 </li><li>继1小时，冲洗的幻灯片在DDH 2 O，除去封闭缓冲任何多余的痕迹。配置适当的废物容器封闭液。 </li><li>转移幻灯片玻璃染色持有者和甩干。干阵列通过将它们放置在配备有摆动板持有者离心机，在10 xg的转速为5分钟。 </li><li>一旦载玻片是干的，它们可以立即孵育或存储。储存条件是在一个密封的塑料袋-20℃。 </li></ol></li></ol> 2.分析聚糖结合蛋白<ol><li>制备加湿室，其中待杂交的阵列将适合。一个简单的室由耐热盘，在分层蘸用纸巾和一个机架，在其上的幻灯片将休息底部。 </li><li>使用生物素化凝集素的SNA（ 桑布卡黑凝集素）和ECA（ 刺桐cristagalli凝集素）以10微克/毫升+ 2微克/毫升链霉-555染料，在探测缓冲液（PBS + 0.01％吐温-20）。对于HA（A /越南/ 1203年至1204年的H5N1和A / KY / 07 H1N1，在这个例子中），使用20微克的起始浓度/ ml的预络合，如前所述34。简要地说，使HA:鼠标-α-链球菌:山羊-α-鼠标Alexa647混合物在封闭缓冲液（PBS + 3％BSA + 0.01％吐温-20）中，在4:2:1的摩尔比。 注:该阵列探测与外源凝集素以确定聚糖被固定和可检测（ 图1B）。 </li><li>的20微升聚糖结合蛋白 - 抗体混合溶液转移至384孔板中并在冰上孵育30分钟。连续稀释凝集素和HA样品1:1，在其合适的缓冲液，8倍，通过将10微升的样品与10微升缓冲液中。 </li><li>吸管8微升样品到微孔表面和孵育在90分钟密封加湿（100％RH）下室中。 </li><li>通过保持边缘和浸渍他们四次和0.05％吐温的PBS的混合物洗载玻片一次一个，然后四PBS倍，并在去离子H 2 O最后四次运用在封闭步骤中描述的相同的干燥协议，旋阵列干燥在离心机中在10×g下5分钟。 </li></ol> 3.扫描和数据分析 注:聚糖微阵列可以在不同的设置，扫描取决于芯片扫描仪制造商。通过改变激光功率和分辨率，该仪器可以产生与它的动态范围（ 图1C）内尽可能多的信号尽可能的图像。 <ol><li>使用荧光滑动扫描器，并与聚糖结合蛋白孵育后，立即扫描阵列。注意确保幻灯片正确插入扫描仪。 <ol><li>开扫描软件。单击开始以打开该程序。连接到扫描仪:扫描仪→连接或绿色按钮在扫描仪的导航面板菜单。 </li><li>扫描仪打开门自动加载磁带机。放入插槽幻灯片在特定的顺序自动加载机。关闭自动加载磁带机门。氯ICK"读装载机"中的工具导航面板自动检测扫描仪的幻灯片。 </li><li>设置扫描参数:合适的实验（ 如 635激光功率高，检测增益50％）扫描仪→扫描参数。扫描幻灯片:扫描仪→扫描。通过创建一个文件夹来保存扫描图像和名称单独扫描选择路径。做到这一点之前，实际的扫描。重复每个单独的幻灯片。单击确定开始扫描。 </li></ol></li><li>使用扫描仪软件来设置仪器和扫描的幻灯片。设置扫描器迭代扫描在低激光功率（5毫瓦）随的25％，50％和75％的增益设置。 注:在设置中进行测试和优化，但要记住，每次扫描可降低可能由于漂白染料的荧光输出。 <ol><li>开放数据采集和分析软件如MAPIX软件。单击开始以打开该程序。打开扫描图像:文件→打开聊天室年龄。打开GAL文件:分析→开放式网格并选择适当的GAL文件。 </li><li>进入块模式:图像→模式的块移动的网格。使用网格标记到网格设置在滑动的适当位置。调整各个块所必需的印刷阵列相匹配。 </li><li>进入点模式:图像→点模式来调整位置和块内的个别景点的大小。一旦所有的块和斑点调整到位，措施的信号强度通过分析→光度计算。减输出文件。 </li></ol></li><li>一旦载玻片完成扫描，图像分析用于与安装的（ 图 1D - G）的图像处理程序的结合的信号。对于图像分析，加载在扫描TIFF图像一个GAL（数组列表）文件。 注意:GAL文件包含当场布局模式，每个点的位置的标识。数组GAL文件是CR通过使用包含样品的身份和他们在384孔源板位置的制表符分隔文本文件中的打印机软件eated。 </li><li>使用印在芯片格标记/着陆灯要正确放置GAL网格。个别位置可能需要被单独移动，但一个印刷井阵列通常将允许在网格内的块放置自如。下面的网格布局，由软件收集点强度，并保存为制表符分隔文本文件（.txt）。 <ol><li>使用电子表格程序，打开扫描仪​​的输出文件。从打开的文件夹位置适当的宏文件。点击查看→宏→RunMacro。 注意:预写将复制的原始输出数据，删除不必要的行和列，由试样的数据进行排序，计算平均平均信号减去背景值，并在绘制所得值以包含每个图表的电子数据表的工作表六SAMP的莱斯阵列上进行测试。 </li></ol></li></ol>

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C., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+microarray+technologies:+tools+to+survey+host+specificity+of+influenza+viruses.">Glycan microarray technologies: tools to survey host specificity of influenza viruses.</a> Nat Rev Microbiol. 4 (11), 857-864 (2006).</li><li>de Vries, R. 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The authors have nothing to disclose

评估IAV的受体特异性在分析禽流感病毒大流行潜力的重要一步。由病毒唾液酸识别被链接到几个生物性能如从细胞结合和释放。其中知识氨基酸的突变是必要的禽流感病毒实现α2-6结合和跨越物种屏障使大流行的准备。数测定法用于确定受体特异性;但是，所有有其缺点，包括尺寸仅为亲和力，而不是特异性， 反之亦然 。 这里，我们描述基于广泛使用和接受的ELISA，一?...

野生水禽是IAV的自然宿主，但是IAV能够跨越物种屏障，禽类和哺乳动物，包括人类。禽流IAVs认识α2-3连接的唾液酸（禽型受体），而人类病毒结合α2-6连接的唾液酸（人型受体）。为了能够有效复制和人类禽类IAV需要绑定到人型受体1之间传递。 IAVs是基于血清学表征其血凝素（HA）和神经氨酸酶（NA）包膜糖蛋白的抗原性划分。 HA结合到唾液酸，而NA是在病毒的生命周期并切割唾液酸2的另一端的受体破坏酶。所有的人感染病毒，包括H1N1，H2N2和H3N2，有禽源3。在过去的二十年中几个禽类到人类交叉的发生，用H5N1，H7N7，和H7N9是最公知的;豪版本，其他亚型传染人类更零星（H6N1，H7N1，H7N2，H9N2，H10N7，H10N8）4。幸运的是，似乎没有这些病毒已经能够完全适应人型α2-6连接的唾液酸受体5-8。禽流感或其他人畜共患的病毒适应，以适应人类宿主复制和传播可能对人体健康产生破坏性的影响。因此，这些病毒是如何发展结合人型受体将有助于新兴流感病毒全球范围内监视的先验知识。 受体偏好的测定采用不同物种的红细胞首次阐明并保持流感研究者9-12之间的青睐检测。即禽流感病毒识别α2-3唾液酸α2-6连接的唾液酸人类病毒的示威最初是基于使用酶设计包含每个李红细胞血凝化验nkages 13,14。虽然读出是血细胞凝集，一个标准测定为病毒学家，底层聚糖结构没有定义，只有终端联系​​。此外，唾液酸转移酶，用于重新sialylate细胞的有限的可用性，已经限制了使用该测定15-18中。接着，使用连接于聚丙烯酰胺（PAA）或聚谷氨酸（PGA）的结构在基于板的测定法19,20唾液酸化聚糖结构中引入确定受体结合偏好的其他方法。几种变化是在涂覆或聚糖或病毒微量滴定板，其中每一个导致一个健壮的，可靠的和非常敏感的ELISA型测定21-23可能。可替代地，生物素连接的聚糖可以取代的PAA / PGA和可以缀合到链霉抗涂层板2,24。虽然可能需要一些特定的血清，酶联免疫吸附都与PAA标准和几个聚糖容易commercially和非商用（财团为功能糖组学（http://www.functionalglycomics.org））。 聚糖微阵列技术已成为一个宝贵的工具，以确定受体特异性，为多个不同的聚糖发现，并结合到多种不同结构可以在单一实验中25-29评估。 IAV的这些结构的结合提供了更好的理解，即IAV优先识别30-33的聚糖结构。聚糖微阵列需要少量的样本量来进行结合分析，只有使用每点（2 NL）聚糖微量。然而，这些阵列通常仅用于评估聚糖受体特异性。的多种病毒，或血凝素蛋白，在多个浓度范围分析可以望而却步由于所需的幻灯片的数量。此外，迄今为止，没有相对亲合力测定已使用聚糖芳开发射线技术。 结合由聚糖微阵列技术和基于ELISA的测定法的PAA联聚糖的灵敏度，得到低样品要求，我们试图开发一个多井聚糖阵列将允许对具有相似或更好的分辨率高通量分析相比传统的基于ELISA的测定法。同时，我们希望最小化消耗的生物制品和报道的化学品的量。最终的结果是一个小型化的亲合力测定中，专门用于监测IAV特异性开发的，同样适用于评估其他聚糖结合蛋白。使用由聚四氟乙烯面具分为48微孔载玻片，6种不同的聚糖看准每孔6个重复。该芯片平台能提供受体趋势相同绑定看到在宏观ELISA格式几个优点。这些包括（I）中6次重复化合物的印刷，使用最少的样品，与若干行的涂布我呐板，使用每孔100μl; （二）多种不同的化合物在单井，包括控制同时进行分析; （Ⅲ）在培养体积和大规模减少; （IV）使用荧光读出一个较大的动态范围。单个幻灯片可以被计算为等于18倍的96孔板。 用下面的协议，任何实验室能够制造和分析的斑点的微阵列应该能够制造这种小型化的ELISA形式。

<table><tbody><tr><td>NEXTERION&amp;reg; Slide H MPX-48&amp;nbsp;</td><td>Schott</td><td>1091525</td><td>Microwell slides</td></tr><tr><td>ProScanArray Plus&amp;nbsp;</td><td>PerkinElmer</td><td>discontinued</td><td>confocal microarray scanner</td></tr><tr><td>Innoscan 1100AL Scanner/Mapix Software</td><td>Innopsys</td><td>-</td><td>confocal microarray scanner</td></tr><tr><td>MicroGrid II&amp;nbsp;</td><td>Digilab</td><td>-</td><td>microaray printer</td></tr><tr><td>SNA</td><td>Vector Labs</td><td>B-1305&amp;nbsp;</td><td>Plant Lectin</td></tr><tr><td>ECA</td><td>Vector Labs</td><td>B-1145&amp;nbsp;</td><td>Plant Lectin</td></tr><tr><td>Anti-Strep Antibody</td><td>IBA</td><td>2-1509-001&amp;nbsp;</td><td>Anitbody for HA binding</td></tr><tr><td>Anti-Mouse Alexa-647</td><td>Life</td><td>A-21235</td><td>Anitbody for HA binding</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P2287&amp;nbsp;</td><td>detergent</td></tr><tr><td>di-basic Sodium Phosphate</td><td>Sigma</td><td>255793</td><td>printing buffer component</td></tr><tr><td>mono-basic Sodium phosphate</td><td>Sigma</td><td>229903&amp;nbsp;</td><td>printing buffer component</td></tr><tr><td>poly-l-lysine solution</td><td>Sigma</td><td>P8920&amp;nbsp;</td><td>pre-spotting slide component</td></tr><tr><td>sodium hydroxide</td><td>Sigma</td><td>221465</td><td>pre-spotting slide component</td></tr><tr><td>ethanol</td><td>Sigma</td><td>493546</td><td>pre-spotting slide component</td></tr><tr><td>phosphate buffered saline</td><td>Corning</td><td>46-013-CM</td><td>incubation/washing buffer</td></tr><tr><td>SMP4B pins</td><td>Telechem</td><td>SMP4B</td><td>printing pin</td></tr><tr><td>Compressed Nitrogen (Grade5)</td><td>Praxair</td><td>UN1066</td><td>general dusting/drying tool</td></tr><tr><td>Boric Acid</td><td>Sigma</td><td>B6768-500G</td><td>Slide blocking reagent</td></tr><tr><td>ethanolamine</td><td>Sigma</td><td>E9508-500ML</td><td>Slide blocking reagent</td></tr><tr><td>Atto 488</td><td>AttoTec</td><td>AD 488-91</td><td>Gridmarker on array</td></tr><tr><td>PAA-LNLN</td><td>Consortium for Functional Glycomics</td><td>PA368</td><td>Spotted glycans</td></tr><tr><td>PAA-3SLNLN</td><td>Consortium for Functional Glycomics</td><td>PA362</td><td>Spotted glycans</td></tr><tr><td>PAA-6SLNLN</td><td>Consortium for Functional Glycomics</td><td>PA343</td><td>Spotted glycans</td></tr><tr><td>LNLN</td><td>Consortium for Functional Glycomics</td><td>Te98</td><td>Spotted glycans</td></tr><tr><td>3SLNLN</td><td>Consortium for Functional Glycomics</td><td>Te175</td><td>Spotted glycans</td></tr><tr><td>6SLNLN</td><td>Consortium for Functional Glycomics</td><td>Te176</td><td>Spotted glycans</td></tr><tr><td>384-well microtiter plate</td><td>Matrix TechCorp</td><td>4361</td><td>Printing plate</td></tr><tr><td>VWR lab marker</td><td>VWR</td><td>52877-150</td><td>Slide Numbering</td></tr><tr><td>Wheaton slide staining dish</td><td>Sigma</td><td>Z103969-1EA</td><td>Blocking and Drying</td></tr></tbody></table>

a miniaturized glycan microarray assay for assessing avidity and specificity of influenza a virus hemagglutinins

A型流感病毒（IAV）血细胞凝集素识别在细胞表面上的唾液酸作为功能​​性受体来获得进入细胞。野生水禽是IAV的自然宿主，但是可以IAV跨越物种屏障家禽，猪，马和人。禽流感病毒识别由α2-3键（禽型受体）附连到倒数第二个半乳糖，而人类病毒优先识别唾液酸与α2-6键（人型受体）唾液酸。并监控如果禽流感病毒正在适应人型受体，可使用几种方法。用合成唾液酸糖苷的多样库聚糖微阵列越来越多地用于评估受体特异性。然而，这种技术不用于测量亲合力。亲合力的测量一般是通过评估系列稀释血凝素或病毒的结合以吸附到常规聚丙烯96孔板聚糖实现。在该测定中，聚糖与α2-3或α2-6唾液酸连接到生物素并吸附到链霉板，或连接到聚丙烯酰胺（PAA），其直接吸附到塑料。我们通过直接印刷的PAA联唾液酸糖苷及其非PAA联的对应于微井载玻片已显著小型化该测定。这种设置，与在单个幻灯片48阵列，使得能够6聚糖8稀释结合蛋白，询问6个不同的聚糖，包括两个非唾液酸化的控制的同时测定。这相当于在传统的平板测定18倍的96孔板。该聚糖阵列格式降低化合物和生物制品的消费，从而大大提高了工作效率。

打印，扫描和数据分析为确保正确打印，至关重要的是有聚四氟乙烯面罩内的斑点网格，它描绘了在幻灯片上的每个阵列的正确对准。在印刷中，由于聚四氟乙烯涂层的性质，点不能由上MPX幻灯片肉眼看到。注意的是然后在聚-L-赖氨酸包被的玻片预斑点的外观。印刷后直接，各滑动应当由眼对每个斑点化合物是可见的存在，由...

Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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7.9K 次观看.  The Scripps Research Institute. Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

视频: A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

科研

免疫与感染

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

医学

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

使用流感抗原微阵列测量病毒亚型血清抗体的广度

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Source: Kaufmann, L. et al., <a href="https://app.jove.com/v/55833">An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.</a> J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

用于检测针对靶抗原的血清抗体的血凝抑制试验

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://app.jove.com/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

一种测量流感神经氨酸酶抑制抗体滴度的测定法

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...